The ?2A-adrenergic receptor (AR) belongs to the large G protein-coupled receptor (GPCR) superfamily. The many physiological and pharmacological responses mediated by the ?2AAR positions this particular subtype as a potential candidate for pathogenesis and a valuable target for treatment of a variety of diseases including ADHD, hypertension and depression. To date, the endogenous mechanisms whereby ?2AAR functions are regulated at the molecular and cellular levels remain elusive. GPCR interacting partners other than G proteins play pivotal roles in modulating GPCR signaling, and represent an entirely new avenue for drug development. Our previous studies identified spinophilin as an ?2AAR-interacting partner that regulates multiple aspects of ?2AAR trafficking and signaling (i.e. receptor surface retention, internalization, signal activation, desensitization and agonist sensitivity) through antagonizing arrestin functions. Furthermore, the absence of spinophilin alters the intensity and sensitivity of ?2AAR-evoked responses in mouse. The profound in vivo relevance of the ?2AAR-spinophilin interaction motivates us to explore the molecular and cellular mechanisms by which spinophilin regulates ?2AAR trafficking and signaling in native neurons, where both the ?2AAR and spinophilin elicit their major physiological functions. We have generated a novel mouse line in which N-terminal HA tagged ?2AAR expression is driven by the endogenous mouse ?2AAR locus (HA-?2AAR knock-in or ?2AAR HA/HA) and can be detected by HA antibodies. Using this unique knock-in line, in combination with the spinophilin knockout mice (Sp-/-), as well as complementary cellular and molecular strategies, we will test the central hypothesis: spinophilin regulates both the kinetics and agonist-sensitivity of ?2AAR trafficking and signaling in native neurons in order to fine-tune ?2AAR responsiveness. Specific Aim 1 will determine the role of spinophilin in regulation of agonist-induced ?2AAR endocytosis in native neurons. In addition to epinephrine (an endogenous ligand), we will study ?2AAR trafficking induced by clonidine and guanfacine (two ?2-agonists commonly used in the clinic). It would be particularly significant if spinophilin-mediated regulation of the ?2AAR exhibits agonist-bias, which would represent a potential mechanism underlying agonist-selective regulation of receptor trafficking. Specific Aim 2 will determine the role of spinophilin in regulation of ?2AAR - evoked electric (inhibition of Ca2+ currents) and biochemical (activation of ERK) responses in neurons. Specific Aim 3 will determine the role of PKA-mediated phosphorylation of spinophilin in regulation of ?2AAR trafficking and signaling in native neurons. Our newly collected preliminary data demonstrated that PKA phosphorylation of spinophilin disrupts the ?2AAR-spinophilin interaction. We will further determine the functional relevance of this PKA-mediate modulation on ?2AAR trafficking and signaling in native neurons. This revised proposal, significantly improved by our responding to the queries of the reviewers and by the inclusion of considerable additional preliminary data, will advance our understanding of endogenous regulation of the ?2AAR by spinophilin, thus providing new insight for future drug development, and in particular, for the potential design of allosteric agents acting at the ?2AAR to enhance or diminish receptor-spinophilin interactions, depending on the desired functional outcome. PUBLIC HEALTH RELEVANCE G protein-coupled receptors (GPCRs) are members of the largest family of surface receptors and represent the most abundant class of therapeutic targets. The ?2A- adrenergic receptor (?2AAR) subfamily mediates a wide range of critical physiological/pharmacological responses which include lowering blood pressure, evoking sedation, reducing pain perception, and improving working memory. Using genetic models and molecular and cellular strategies, our studies aim to understand how ?2AAR functions are tightly regulated by the interacting protein, spinophilin. Information obtained from our studies will advance our understanding of endogenous cellular mechanisms underlying ?2AAR functions, and thus provide novel insight into therapeutic strategies aimed at the treatment of attention deficit and hyperactivity disorder (ADHD), depression, hypertension and other clinical settings where use of ?2AAR-agonists may be warranted.